Plated Hepatocyte Relay Assay (PHRA) for the estimation of intrinsic... metabolite profile of slowly metabolized compounds

Plated Hepatocyte Relay Assay (PHRA) for the estimation of intrinsic... metabolite profile of slowly metabolized compounds

Plated Hepatocyte Relay Assay (PHRA) for the estimation of intrinsic hepatic clearance and
metabolite profile of slowly metabolized compounds
In Vitro ADMET
Laboratories, LLC
Albert P. Li,* Chi-Chi Peng, Utkarsh Doshi, Chandra Prakash, In Vitro ADMET Laboratories (UD, APL) and Biogen Inc. (CCP, CP)
On day 2, the incubation medium from day 1 is transferred to a new culture
of human hepatocytes prepared from the same lots used on day 1 followed
by another 24 hr of incubation (1st relay). The process is repeated on days
3 (2nd relay), 4 (3rd relay), and 5 (4th relay). With this novel relay method,
a chemical can be evaluated for metabolism by human hepatocytes for up
to 5 days.
Data Analysis:
Compounds’ parent accurate mass was used to do quantification.
Peak area was normalized by internal standard.
Concentration after each relay was corrected for loss of drug in plated
hepatocytes post-pooling according to Li Di’s paper.
Percent remaining at each time points were compared to that of Time 0.
Following equations were used to calculate the CLint and Clh.
CLint = ln2*(1/T1/2 min)*(mL incubation/0.5 Mcells)*(120 M cells/g
liver)*(21g liver/kg) = ml/min/kg
CLh=Q*fu*Clint/(Q+(fu*Clint), (fu=1;human Q (ml/min/kg): 21).
3) The supernatants (150 µL) were transferred to a new plate and injected to HPLC coupled with
AB Sciex triple TOF 5600 system
3) Incubate for 24 h
Day 2 (1st Relay)
HPLC system
1) Thaw fresh vials and plate hepatocytes to prepare the first relay plate (Plate 2).
2) T= 24 h: Pool the media containing the same chemical in all the wells in Plate 1. Remove
aliquots for LCMS analysis.
HPLC column
Waters Acquity System
Time
Flow Rate
Waters Corp.
(Marlborough, MA)
(min)
(mL/min)
0
0.45
95
5
0.2
0.45
95
5
3
0.45
10
90
3.5
0.45
10
90
3.6
0.45
95
5
4
0.45
95
5
Acquity, UPLC BEH
C18 1.7 µm, 2.1 x 50 mm
Waters Corp.
3) Replace medium in Plate 2 with the pooled medium for the 1st Relay (T= 24 to 48 h).
Incubate for 24 h.
4) Repeat procedures for Days 3 (2nd Relay; T=48 to 72 hr). 4 (3rd Relay; T=72 to 96 h) and 5
(4th Relay: T= 96 to 120 h)
Column
temperature
55°C
Mobile Phases:
A. 0.1% formic acid (ESI +) or 10 mM
ammonium acetate, pH 6 (ESI -)
B. Acetonitrile (ACN)
A (%) B (%)
Results
Slope: -0.1464 ± 0.007113
4
3
2
1
0
0
10
Time (hrs)
20
30
Clozapine
5.0
Slope: -0.03504 ± 0.004935
4.5
4.0
3.5
3.0
2.5
6
4
0
20
Time (hrs)
40
60
Glimepiride
Slope: -0.2179 ± 0.02527
2
0
-2
10
20
Time (hrs)
30
5.0
Ln(% of Parent Remaining)
5
Meloxicam
Slope: -0.01527 ± 0.0009626
4.5
4.0
3.5
3.0
2.5
2.0
0
50
100
Time (hrs)
150
3
2
1
0
0
50
100
Time (hrs)
150
Riluzole
6
Slope: -0.09326 ± 0.003895
4
2
0
-2
20
40
60
0
Time (hrs)
CLh
(ml/min/kg)
in vitro
Clnonrenal, blood
(ml/min/kg)
in vivo
4
6
8
10
Risperidone
12.3
7.75
5.16
Clozapine
2.94
2.58
2.9
Glimepiride
18.3
9.8
1.12
Meloxicam
1.29
1.2
0.12
Tolbutamide
2.15
1.96
0.31
Riluzole
7.84
5.7
2.05
Ziprasidone
10.2
6.9
CL: 11.7
Diclofenac
49.8
14.8
7.67
Ibuprofen
25.7
11.6
1.5
-2
Ziprasidone
5
Slope: -0.1213 ± 0.001862
4
3
2
1
0
0
10
Time (hrs)
20
30
Ibuprofen
4
Slope: -0.3064 ± 0.02053
3
2
1
0
2
4
6
Time (hrs)
8
Clozapine, riluzole only first 48 hrs data
Ibuprofen in “–” mode, no IS normalization
Incubation volume: 0.1mL
10
Conclusions
Summary
The Plated Hepatocyte Relay Assay (PHRA) was validated with 10 model
drugs with in vivo hepatic intrinsic clearance ranged from 0.081
mL/min/kg (warfarin; slowly metabolized) to 7.67 mL/min/kg (diclofenac;
rapidly metabolized).
Linear time-dependent parent disappearance was observed, with the
calculated hepatic clearance values falling comparable to the known in
vivo values.
2
5
0
Compound
CLint (ml/min/kg)
in vitro
2
Time (hrs)
Slope: -0.02568 ± 0.0008154
4
Slope: -0.5954 ± 0.04002
4
Tolbutamide
5
Diclofenac
6
Ln(% of Parent Remaining)
Risperidone
Ln(% of Parent Remaining)
Methods:
Plated Hepatocyte Relay Assay (PHRA): The relay assay consists of firstly
culturing plateable cryopreserved human hepatocytes (pooled from four
donors) on day 1 for approximately 4 hrs to allow attachment, followed by
incubation with the chemicals to be evaluated for a 24 hr. period.
2) Samples were vortexed and centrifuged at 3220 g (Eppendorf centrifuge) for 10 minutes.
Ln(% of Parent Remaining)
Materials:
Plateable Cryopreserved Human Hepatocytes (IVAL, Columbia, MD): Pool of
hepatocytes from four donors – HH1007, HH1023, HH1031, and HH1036
Collagen Coated 96 well plates (CellAffix™; APSciences Inc., Columbia, MD)
Universal Cryopreservation Recovery Medium (UCRM; IVAL) for the
recovery of cryopreserved hepatocytes
Universal Primary Cell Plating Medium (UPCM; IVAL) for the initial plating of
the hepatocytes
Hepatocyte Incubation Medium (HQM; IVAL) for assay
2) T= 0 hr: Replace medium to that containing chemicals to be evaluated (remove an aliquot as
no hepatocyte control)
Ln(% of Parent Remaining)
Materials & Methods
1) Supernatants (200 µL) were thawed at room temperature.
Ln(% of Parent Remaining)
To overcome this challenge, a relay long term metabolism assay has been
developed with primary cultured hepatocytes.
Sample Treatment and LC/MS Analysis
1) Thaw and plate human hepatocytes (Plate 1) and allow 4 h for attachment
Ln(% of Parent Remaining)
The evaluation of metabolic fate and estimation of in vivo intrinsic
clearance of these “slowly metabolized” compounds is a technical
challenge, as in general, little metabolism can be observed using the
routine short-term incubations with human liver microsomes, S9, or human
hepatocyte suspensions.
Day 1
Ln(% of Parent Remaining)
The current practice of metabolic stability screening of new chemical
entities (NCE) has led to the collection of chemicals that are resistant to
metabolism by in vitro metabolic experimental system such as human liver
microsomes and human hepatocytes.
Procedures
Ln(% of Parent Remaining)
Introduction
Introduction
The results suggest that PHRA can be used for the estimation of in vivo
hepatic clearance of slowly metabolized compounds that cannot be readily
evaluated using routine methods. The assay can also be used for the
metabolite profiling and phenotyping of slowly metabolized compounds.
*Corresponding author: Albert P. Li (lialbert@invitroadmet.com)
ISSX - JSSX 2014